Guan et al. BMC Ophthalmology (2017) 17:173 DOI 10.1186/s12886-017-0567-3

RESEARCH ARTICLE Open Access Analysis of the VSX1 in sporadic keratoconus patients from China Tao Guan1†, Xue Wang2†, Li-Bin Zheng3, Hai-Jian Wu1 and Yu-Feng Yao3*

Abstract Background: Keratoconus normally presents as a sporadic disease. Although different studies have found sequence variants of the visual system 1 (VSX1) gene associated with keratoconus in humans, no research has detected such variants in sporadic keratoconus patients from China. To investigate the possibility of VSX1 being a candidate susceptibility gene for Chinese patients with sporadic keratoconus, we performed sequence screening of this gene in such patients. Methods: Whole DNA was obtained from the leukocytes in the peripheral venous blood of 50 patients with sporadic keratoconus and 50 control subjects without this ocular disorder. Polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing technology were used to detect sequence variation in the five exons and splicing regions of the introns of the VSX1 gene. The sequencing results were analyzed using DNAstar software. Results: One novel missense heterozygous sequence variant (p.Arg131Pro) was found in the first exon of the VSX1 gene in one keratoconus patient. Another heterozygous sequence variant (p.Gly160Val) in the second exon was found in two keratoconus patients. These variants were not detected in the control subjects. In the third intron of the VSX1 gene, c.8326G > A nucleotide substitution (including heterozygous and homozygous change) was also discovered. The frequency of this variation did not differ significantly between patients and controls, it should belong to single-nucleotide polymorphism of the VSX1 gene. Bioinformatic analysis also predicted that one missense sequence variation (p.Arg131Pro) may not cause a pathogenic change. Conclusions: In this study, we added one novel missense sequence variation (p.Arg131Pro) in the coding region of the VSX1 gene to the range of VSX1 coding region variations observed in patients with sporadic keratoconus from China. Our work suggests that VSX1 sequence variants might be involved in the pathogenesis of sporadic keratoconus, but their precise role in disease causation requires further investigation. Keywords: Sporadic keratoconus, VSX1 gene, Sequence variant, Single-nucleotide polymorphism

Background [3, 4]. The pathological features of keratoconus include Keratoconus is a congenital disease involving noninflam- decreased corneal collagen or the abnormal distribution matory corneal ectasia. Its prevalence is estimated to be of collagen fiber, which reduce the mechanical resistance 1/2000 in the general population, and the incidence ratio of the cornea and therefore cause the center of the between males and females is 1.7:1 [1, 2]. Although most cornea to protrude, leading to a thin conical shape. Slit keratoconus cases are sporadic, about 6–10% of cases lamp microscope examinations of keratoconus patients have been reported to have a positive family history. have demonstrated Vogt’s line, Fleischer’s ring, and cor- Keratoconus can exhibit dominant or recessive inherit- neal scarring [5–7]. The onset of the disease occurs during ance; with autosomal dominant inheritance, the disease puberty, involving both eyes, and with highly irregular exhibits variable phenotypes with incomplete penetrance astigmatism. In the late phase, patients show acute corneal edema and scar formation, along with significant vision * Correspondence: [email protected] loss. Although several treatment modalities are available, †Equal contributors 3 severe keratoconus remains an indication for corneal Department of Ophthalmology, Sir Run Run Shaw Hospital, Zhejiang – University School of Medicine, Hangzhou 310016, China transplantation [8 10]. Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guan et al. BMC Ophthalmology (2017) 17:173 Page 2 of 7

Genome-wide linkage analyses have identified several members and identifying the patient as an isolated case chromosomal loci and that may be related to kera- of keratoconus. Another 50 healthy people who under- toconus; some of these were eventually ruled out, while went detailed ocular and ophthalmological examinations for others, an association with this disease could not be that ruled out any ophthalmic diseases were selected as confirmed. One of the candidate genes strongly impli- a control group to determine whether the detected vari- cated in keratoconus is visual system homeobox gene 1 ation was SNP of VSX1 gene. The control subjects in- (VSX1) localized to 20 p11–q11 [11–14]. cluded 31 males and 19 females, aged from 20 to The VSX1 gene encodes a paired-like homeodomain 36 years old. The age and gender generally matched be- . It is associated with eye develop- tween the two groups and systemic physical examina- ment and was first isolated and cloned from a cDNA li- tions were performed in both groups to rule out other brary of the retina of adult goldfish. In the human body, concurrent diseases. This study followed the Declaration the VSX1 gene is expressed in the embryonic craniofa- of Helsinki and was reviewed by the ethics committee of cial region, the granular layer of adult retina, and corneal Taizhou Municipal Hospital. Written informed consent tissue. The human VSX1 gene has five exons and en- was obtained from all of the subjects in this study. codes a 365-amino-acid with a homeobox DNA binding domain and a CVC domain, being highly con- Genetic study served among vertebrates. Barbaco et al. [15] reported Three milliliters of peripheral venous blood was ex- that the VSX1 gene was activated in corneal stroma dur- tracted from the subjects and stored in EDTA anticoagu- ing the process of corneal wound repair in vitro and in lation tubes. Genomic DNA was isolated from blood vivo, accompanied by the transformation of corneal stro- leukocytes using the Dzup (Blood) Genomic DNA Isola- mal cells into myofibroblasts. These results implied that tion Reagent (B518205; Sangon Biotech Co., Ltd., Shang- VSX1 gene variants may play an important role in the hai, China), in accordance with the manufacturer’s development of keratoconus. Several variants of the standard methods. VSX1 gene [16–19] have been reported from various Based on previous studies [20, 21] and the DNA se- parts of the world, but a definitive pathogenic role of quence of the human VSX1 gene from the NCBI data- these variants in keratoconus has not yet been estab- base, primers were designed for the five exons of this lished because segregation of these variants was also gene. These primers were synthesized by Sangon Biotech seen in some unaffected individuals. Co., Ltd. (Shanghai, China). Sequences of the primers To our knowledge, this is the first study to perform are listed in Table 1. screening for variants of the VSX1 gene in the Chinese The HBP 220 PCR cycler (HYBAID, England) was population and to explore their role in keratoconus. used for a 50-μL PCR reaction system containing 200 ng Nucleotide variations in the five exons and the splicing of DNA template, 5 μL of 10× buffer solution, regions of introns of the VSX1 gene were examined by 1.5 mmol/L MgCl2, 0.25 μmol/L primers, 100 μmol/L of polymerase chain reaction single-strand conformation each dNTP, and 2 U of Taq DNA polymerase (Sangon polymorphism (PCR-SSCP) analysis and direct DNA se- Biotech Co., Ltd., Shanghai, China). The PCR procedure quencing in 50 Chinese patients with keratoconus, was as follows: 94 °C for 5 min; 35 amplification cycles which were compared with the sequences in 50 controls. of denaturation at 94 °C for 45 s, annealing for 45 s, and extension at 72 °C for 50 s; and finally extension at 72 ° Methods C for 10 min. PCR products were examined by 2% agar- Subjects and clinical examination ose gel electrophoresis and EB (Ethidium Bromide) All sporadic keratoconus patients and control subjects staining. The annealing temperatures of each exon and were identified at the Department of Ophthalmology, the fragment sizes of PCR products are listed in Table 1. Taizhou Municipal Hospital. A total of 50 patients were An 8% (49:1) nondenaturing polyacrylamide gel with diagnosed with sporadic keratoconus during the period 5% glycerol was used for SSCP analysis and silver stain- of March 2013 to July 2015, including 29 males and 21 ing. Here, 5-μL PCR products mixed with sample buffer females, aged from 15 to 35 years old. The diagnosis was (90% formamide, 0.05% bromophenol blue, and 0.05% based on symptoms and the results of slit lamp micro- xylene blue) were processed for denaturation at 95 °C scope examinations; the diagnostic features included for 10 min, placed in an ice bath, and then subjected to progressive curvature of the anterior corneal surface electrophoresis (100 V) at room temperature for 5 to steepening, corneal thinning in the same region, 6 h (when xylene blue reached the bottom of the gel). Fleischer’s ring, and Vogt’s stripes, combined with the Then, the gel was fixed in 10% ethanol for 5 min and corneal surface topography and a caudal surface of the soaked in 1% silver nitrate for 5 min, followed by stain- cornea thicker than 20 μm. Patients were considered as ing in 0.012 mol/L silver nitrate and 0.05% formaldehyde sporadic cases after examining the immediate family for 10 min. Next, the gel was processed in 0.28 mol/L Guan et al. BMC Ophthalmology (2017) 17:173 Page 3 of 7

Table 1 List of primers and PCR amplification conditions used for VSX1 gene amplification Exon Sequence of primers (5′-3′) Annealing Product temperature (°C) length (bp) 1 CAGCTGATTGGAGCCCTTC(sense) 62 599 CTCAGAGCCTAGGGGACAGG(antisense) 2 CTTAAGTACCCAAGAGGTTCATAACT(sense) 62 269 GAAACCACTGGGCCTGCTATCAT(antisense) 3 AAGCAGGCACGGTGGTCCTTA(sense) 63 293 AAGGGACTGCTGATTGGCTCACT(antisense) Fig. 1 Electrophoresis pattern of PCR products of the VSX1 gene. M: 4 GCTCGGGAGAGAAGATCC(sense) 62 399 DNA markers, 1: first exon (599 bp), 2: second exon (269 bp), 3: third TCAGTAAACTGACGTTGCTTG(antisense) exon (293 bp), 4: fourth exon (399 bp), 5: fifth exon (495 bp) 5 GGAAATTTACTTCATTGCTGAATTT(sense) 60 495 TGGCATTGCATTTTATCTTGACA(antisense) analysis and silver staining, four types of abnormal band- ing pattern were clearly shown in the electrophoresis sodium carbonate, 0.1% formaldehyde, and 0.1 g/L gels, suggesting the presence of four sequence variants thiosulfate sodium for colorization, which was stopped in the five exons of the VSX1 gene. by immersion in 10% acetic acid. DNA with an abnormal banding pattern in electro- DNA sequencing results phoresis was detected by SSCP and silver staining ana- The 50 sporadic keratoconus patients and 50 control lysis. Its PCR product was then purified using isopropyl subjects recruited for this study underwent screening of alcohol and bidirectionally screened to identify the se- the entire coding region and exon–intron junctions of quence variant using the ABI3730XL DNA Analyzer the VSX1 gene by direct sequencing of DNA. The DNA (Sangon Biotech Co., Ltd., Shanghai, China). Nucleotide sequencing results confirmed the abnormal banding pat- sequences were then compared with the published VSX1 terns found in the SSCP analysis and silver staining as cDNA sequence (GenBank NM_014588). described above. The results revealed four nucleotide sequence variants of the VSX1 gene in Chinese Bioinformatic analysis sporadic keratoconus patients, including two missense The in silico prediction programs Polyphen-2 (http:// sequence variants and one SNP change (heterozygous genetics.bwh.harvard.edu/pph2/) and PROVEAN (http:// and homozygous) (Table 2). provean.jcvi.org/index.php) were used to predict the The sequence variant c.5427G > C was found in the first effect of the VSX1 gene sequence variants on protein exon of the VSX1 gene in one case of sporadic keratoconus. structure and function [22–24]. This involved a change in the codon from CGC to CCC, leading to a missense substitution of arginine to proline at Statistics codon 131 (p.Arg131Pro sequence variant). p.Arg131Pro To determine statistically significant differences between was not detected in any control subjects (Fig. 2) and had the groups, data analysis was performed using SPSS ver- not previously been identified. We registered it in a public sion 18.0. Continuous data are displayed here as mean ± database at https://www.ncbi.nlm.nih.gov/projects/SNP/ standard deviation. Differences between two groups were (ID: rs267597889). Using the PolyPhen-2 tool, p.Arg131Pro examined by t-test. Repeated measure ANOVA was used was assessed as being benign, in terms of its phenotypic ef- to analyze the differences among multiple groups. Values fects, with a score of 0.344; using the PROVEAN tool, it of P < 0.05 were considered statistically significant. wasassessedasbeingneutral,withascoreof−0.048. Both tools predicted that the replaced amino acid would have no Results adverse effects and would be tolerated. PCR-SSCP results The other missense sequence variant c.7672G > T was All exons of the VSX1 gene in sporadic keratoconus pa- found in the second exon of the VSX1 gene in two cases tients were amplified by PCR. The PCR products showed of sporadic keratoconus. This involves a change in high specificity and efficiency in amplification by 2% codon 160 from GGC to GTC, leading to a missense agarose gel electrophoresis, with a single band corre- substitution of glycine to valine (p.Gly160Val sequence sponding to the correct fragment length (Fig. 1). This variant) (Fig. 3). No similar change was found in any of suggested that there were no sequence variants with a the 50 normal controls. large insertion or deletion in the five exons of the VSX1 In the third intron of the VSX1 gene, the substitution gene in the sporadic keratoconus patients. By SSCP of G > A at nucleotide position 8326 (c.8326G > A) was Guan et al. BMC Ophthalmology (2017) 17:173 Page 4 of 7

Table 2 List of sequence variants of the VSX1 gene in Chinese sporadic keratoconus patients Nucleotide change Amino acid change Change site Change type Keratoconus (n = 50) Controls (n = 50) c.5427G > C p.Arg131Pro First exon Heterozygous missense variant 1 (2.0%) 0 (0.0%) c.7672G > T p.Gly160Val Second exon Heterozygous missense variant 2 (4.0%) 0 (0.0%) c.8326G > A Noncoding region Third intron Heterozygous SNP variant 3 (6.0%) 4 (8.0%) c.8326G > A Noncoding region Third intron Homozygous SNP variant 2 (4.0%) 1 (2.0%) identified, which was present in heterozygous form in research, VSX1 is the only gene indicated as a vital three cases of sporadic keratoconus and in four controls genetic factor in determining the keratoconus [29]. (Fig. 4). It was also present in homozygous form in two Verma et al. ruled out the involvement of the VSX1 gene sporadic keratoconus patients and one control (Fig. 5). in South Indian patients with sporadic keratoconus [30]. There was no significant difference in the incidence of However, Shetty et al. found that p.Leu268His, a novel c.8326G > A between the two groups. missense variation in the coding region of VSX1, might be involved in the pathogenesis of sporadic keratoconus in Discussion Indians [22]. These earlier studies indicated the need for Keratoconus is a complex condition with a multi- further study on the relationship between VSX1 patho- factorial etiology that normally occurs as a sporadic genic variations and sporadic keratoconus in different disorder [25]. Recent studies have revealed the in- subsets of the keratoconus-affected population. Shetty et volvement of various genes in its development, such al. also summarized the VSX1 coding variants that are as- as VSX1, SOD1, COL6A1, TGFBI, DOCK9, STK24, sociated with keratoconus in different ethnic groups, but and IPO5 [26–28]. Despite elucidate the etiology and the Chinese were not included in this work (Table 3) [16– the disease progression were conducted by numerous 19, 22, 29, 31–38]. In the present study, PCR-SSCP and direct DNA sequencing techniques were used to examine

Fig. 2 The p.Arg131Pro variant of the VSX1 gene. The sequence of a Fig. 3 The p.Gly160Val variant of the VSX1 gene. The sequence of a normal control (a) included the nucleotides CGC at codon 131 in normal control (a) included the nucleotides GGC at codon 160 in exon 1 of VSX1. The missense variant sequence (b) showed a G to C the second exon of VSX1. The missense variant sequence (b) (c. 5427 G > C) heterozygous transition at the second nucleotide showed a G to T (c. 7672 G > T) heterozygous transition at the position in this codon (arrow), which results in a change from second nucleotide position in this codon (arrow), which results in a arginine (CGC) to proline (CCC) (p.Arg131Pro) change from glycine (GGC) to valine (GTC) (p.Gly160Val) Guan et al. BMC Ophthalmology (2017) 17:173 Page 5 of 7

Fig. 5 The c.8326G > A homozygous change of the VSX1 gene. The Fig. 4 The c.8326G > A heterozygous change of the VSX1 gene. The sequence of a normal control (a) included the nucleotide G at sequence of a normal control (a) included the nucleotide G at position 8326 in the third intron of VSX1. The variant sequence (b) position 8326 in the third intron of VSX1. The variant sequence (b) showed a G to A homozygous transition (arrow), which results in a showed a G to A heterozygous transition (arrow), which results in a c.8326G > A SNP change c.8326G > A SNP change

Table 3 Summary of VSX1 coding variants identified in patients with keratoconus [22] Coding variants Clinical significance Phenotype Unrelated Controls Ethnic groups References p.Leu 17 Pro Pathogenica Keratoconus – Italian [17] p.Leu 17 Val Nonpathogenic Keratoconus + Korean [31] p. Pro 58 Leu Pathogenica, b Keratoconus – Caucasian [32] p. Leu 159 Met Unknown Keratoconus – Caucasian [16, 18] p. Asn 151 Ser Pathogenica Keratoconus – Korean [33] p. Gly 160 Val Nonpathogenic Keratoconus + Korean [31, 33] p. Val 199 Leu Nonpathogenic Keratoconus + Korean [31] p. Arg 166 Trp Unknown Keratoconus + Caucasian, Iranian [16, 34] p. Gln 175 His Unknown Keratoconus – Indian [35] p. Arg 217 His Nonpathogenic Keratoconus + Indian, Pakistan, European [19, 36] p. Gly 239 Arg Pathogenica, b Keratoconus – Italian [29] p. His 244 Arg Unknown Keratoconus + Caucasian, Iranian [16, 18, 34, 37] p. Ser 251 Thr Unknown Keratoconus – Indian [22] p. Pro 247 Arg Nonpathogenic Keratoconus + Italian [16, 17, 38] p. Leu 268 His Pathogenica, b Keratoconus – Indian [22] Coding variants of the VSX1 gene have been reported in studies based on an original reporta and bioinformatic predictionsb. The symbols “+” and “−” represent present and absent, respectively Guan et al. BMC Ophthalmology (2017) 17:173 Page 6 of 7

nucleotide variations in the five exons and splicing regions arginine with proline) has not been reported previously, so of introns of the VSX1 gene in 50 unrelated Chinese this work adds to the range of variants in the VSX1 gene patients with keratoconus, which were compared with the and suggests the role of this novel variant in keratoconus. sequences in 50 controls. The results showed the presence However, predictions made using the PolyPhen-2 and of four nucleotide variations in sporadic keratoconus PROVEAN tools suggested that this variant would not patients, consisting of a missense p.Arg131Pro sequence have detrimental phenotypic effects. This is explained by variant (heterozygous), a missense p.Gly160Val sequence the fact that the 131st amino acid arginine is located in a variant (heterozygous), and nucleotide c.8326G > A noncritical open reading frame region of the VSX1 gene, sequence variation (in both heterozygous and homozy- so its character is not particularly important and it has not gous forms). p.Gly160Val was located in the second been conserved among the gene’s orthologs. The presence exon of the VSX1 gene. In 2008, Mok et al. [33] first re- of p.Arg131Pro in just 2% of the patients in this study ported that p.Gly160Val variation was found in 13 out reinforces the assertion that this is a minor gene, but its of 249 cases (5.3%) of sporadic keratoconus in South exact role during development needs to be clarified. Korea, being located in the homeobox DNA binding domain of VSX1 gene coding regions and involving a Conclusions polar amino acid being replaced by a neutral one. This In summary, the p.Arg131Pro, p.Gly160Val, and VSX1 mayaffectthebindingofthe protein to DNA c.8326G > A variations in the VSX1 gene were identified and modify the transcription rate. Therefore, it was be- for the first time in Chinese patients with sporadic VSX1 lieved that p.Gly160Val of the gene increases the keratoconus. We added one novel missense sequence risk of the onset of keratoconus. In this study, we iden- variation (p.Arg131Pro) in the coding region of the tified p.Gly160Val in two cases of 50 sporadic keratoco- VSX1 gene to the existing range of identified VSX1 vari- nus patients, the sequence variant rate was 4.0%, but ations. However, it was predicted that this novel variant was absent in the control group. The findings suggested may not cause a pathogenic change. Large-scale studies that p.Gly160Val may be the cause of two cases of spor- recruiting more patients are required to obtain a deeper adic keratoconus among these Chinese subjects. How- understanding of the genetic mechanisms underlying the ever, further biophysical studies are necessary to pathogenesis of keratoconus. evaluate the precise molecular mechanism behind the effects of this variant form of VSX1. Abbreviations PCR-SSCP: Polymerase chain reaction single-strand conformation polymorph- The c.8326G > A sequence variation occurred in the VSX1 VSX1 ism; SNP: Single-nucleotide polymorphism; : Visual system homeobox third intron of the gene. Abu-Amero et al. [25] gene 1 detected c.8326G > A heterozygous variation in the Saudi Arabian population. They identified this variation Acknowledgments in keratoconus patients as well as in controls, with no We are grateful to the patients and their families for participating in this study. We also thank staff at the Second Hospital of Ningbo Yingzhou for difference in its incidence between the two groups. This their help and support. variation is located within an intron and does not cause a change in the amino acid sequence encoded by the Funding VXS1 The study was supported by the Medical Scientific Research Foundation of gene. Therefore, they considered that c.8326G > A Zhejiang Province, China (Grant No. 2013KYA233). variation belonged to SNP change of VSX1 gene. In our study, the c.8326G > A variant of the VSX1 gene was Availability of data and materials identified in both heterozygous and homozygous form, The datasets supporting the conclusions of this article are included within the article. The datasets used and analyses performed during the current and it did not differ significantly in its incidence between study are available from the corresponding author or the first author on the two groups. Overall, our results demonstrate the exist- reasonable request, but confidential patient data will not be shared. ence of SNPs in the VSX1 gene in the Chinese population, and suggest that sporadic keratoconus patients from Authors’ contributions YFY conceived and designed the study. TG analyzed and interpreted the differentethnicgroupsmayhavethesamepathogenesis. patient data, and wrote the manuscript. XW performed a critical review and In the present study, we identified one novel heterozy- helped to revise the manuscript. LBZ used bioinformatic tools to predict the gous variant c.5427G > C (p.Arg131Pro; rs267597889) of effects of the . HJW performed statistical analysis and helped to collect the data. All authors read and approved the final manuscript. the VSX1 gene in a 20-year-old affected male, which was absent from all 50 controls. This patient had a history of Ethics approval and consent to participate keratoconus lasting more than 5 years. The slit lamp This study followed the Declaration of Helsinki and was reviewed by the microscope examination showed conical protrusion ethics committee of Taizhou Municipal Hospital. Written informed consent was obtained from all of the subjects in this study. (opacity at the top) of the cornea in both eyes, with corneal thinning and a vertical linear scar, as well as complete Consent for publication Fleischer’s ring. p.Arg131Pro (replacing the amino acid Not applicable. Guan et al. BMC Ophthalmology (2017) 17:173 Page 7 of 7

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Invest Ophthalmol Vis Sci. 2009;50:1081–6. 13. Burdon KP, Coster DJ, Charlesworth JC, et al. Apparent autosomal dominant keratoconus in a large Australian pedigree accounted for by digenic inheritance of two novel loci. Hum Genet. 2008;124:379–86. 14. Abu-Amero KK, Kondkar AA, Azad TA, et al. Keratoconus is associated with increased copy number of mitochondrial DNA. Mol Vis. 2014;20:1203–8. 15. Barbaro V, Di Iorio E, Ferrari S, et al. Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing. Invest Ophthalmol Vis Sci. 2006;47:5243–50. 16. Heon E, Greenberg A, Kopp KK, et al. VSX1: a gene for posterior polymorphous dystrophy and keratoconus. Hum Mol Genet. 2002;11:1029–36. 17. Bisceglia L, Ciaschetti M, De Bonis P, et al. VSX1 mutational analysis in a Submit your next manuscript to BioMed Central series of Italian patients affected by keratoconus: detection of a novel mutation. Invest Ophthalmol Vis Sci. 2005;46:39–45. and we will help you at every step: 18. Tang YG, Picornell Y, Su X, et al. Three VSX1 gene mutations, L159M, R166W, and H244R, are not associated with keratoconus. Cornea. 2008;27:189–92. • We accept pre-submission inquiries 19. Tanwar M, Kumar M, Nayak B, et al. VSX1 gene analysis in keratoconus. • Our selector tool helps you to find the most relevant journal Mol Vis. 2010;16:2395–401. • We provide round the customer support 20. Paliwal P, Singh A, Tandon R, et al. A novel VSX1 mutation identified in an individual with keratoconus in India. Mol Vis. 2009;15:2475–9. • Convenient online submission 21. Stabuc-Silih M, Strazisar M, Hawlina M, et al. Absence of pathogenic • Thorough peer review mutations in VSX1 and SOD1 genes in patients with keratoconus. Cornea. • Inclusion in PubMed and all major indexing services 2010;29:172–6. 22. Shetty R, Nuijts RM, Nanaiah SG, et al. Two novel missense substitutions in • Maximum visibility for your research the VSX1 gene: clinical and genetic analysis of families with Keratoconus from India. BMC Med Genet. 2015;16:33–42. Submit your manuscript at www.biomedcentral.com/submit